Control of white mold (Sclerotinia sclerotiorum) through plant-mediated RNA interference

The causative agent of white mold, Sclerotinia sclerotiorum, is capable of infecting over 600 plant species and is responsible for significant crop losses across the globe. Control is currently dependent on broad-spectrum chemical agents that can negatively impact the agroecological environment, presenting a need to develop alternative control measures. In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing hairpin (hp)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 compared to wild-type Col-0. To better understand how HIGS influences host–pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 directly at the site of S. sclerotiorum infection. RNA sequencing data reveals enrichment of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) pathway, as well as transcription factors predicted to regulate plant immunity. Using RT-qPCR, we identified predicted interacting partners of ABHYDROLASE-3 in the polyamine synthesis pathway of S. sclerotiorum that demonstrate co-reduction with ABHYDROLASE-3 transcript levels during infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provide insight into the role of ABHYDROLASE-3 in the A. thaliana–S. sclerotiorum pathosystem.

insight into the control S. sclerotiorum prior to systemic infection. Further, while ABHYDROLASE-3 silencing has demonstrated reduced disease severity against S. sclerotiorum using SIGS and HIGS technology, its role in pathogenesis is still largely unknown and identification of interacting partners and pathways may provide new candidates to target through RNAi.
Plants activate innate immunity pathways in response to pathogen attack and are dependent on the recognition of specific pathogen-derived molecules 19 . These pathways are commonly separated into necrotrophic and biotrophic responses and demonstrate distinct modes of pathogen detection. For example, necrotrophic pathogens are detected through the presence of pathogen and damage associated molecular patterns (PAMPs/ DAMPs) via pattern recognition receptors (PRRs) in pattern-triggered immunity (PTI), while in contrast, biotrophic pathogens are detected via intracellular NBS-LRR proteins detecting secreted effector molecules in effector-triggered immunity (ETI) 19 . Activation of these recognition pathways induces specific defense hormones which can further activate down-stream defense processes to appropriately respond to pathogen attack. These hormones include jasmonic acid (JA) and ethylene (ET) which can directly activate the induced systemic resistance (ISR) pathway and are associated with promoting physical barriers against necrotrophic pathogens 20 . Other phytohormones like salicylic acid (SA) activate the systemic acquired resistance pathway (SAR) leading to a local hypersensitive response as a defense against biotrophic infection 21,22 . While S. sclerotiorum has been previously categorized as a necrotrophic fungal pathogen, recent reports provide evidence of an early biotrophic phase 23,24 . S. sclerotiorum secretes pathogenicity factors including oxalic acid (OA) and a suite of cell wall degrading enzymes to weaken and break down host cells upon infection 25,26 . During the initial stages of infection, a brief biotrophic phase may occur, where OA and other secreted pathogenicity factors act to suppress host defenses, specifically the SA-mediated SAR pathway 23,24 . The inability of S. sclerotiorum to successfully suppress SAR may lead to increased resistance against S. sclerotiorum infection. Further support for this initial biotrophic phase comes from the identification of secreted effector molecules essential for pathogenicity 27 and intracellular NBS-LRRs necessary for successful defense against S. sclerotiorum 28 . Lastly, synergistic effects between SA-mediated SAR and JA/ET-mediated ISR have been described against necrotrophic infection 29 and that removal of either of these defense hormones will result in an unsuccessful defense response 30 .
In the current study, we engineered A. thaliana expressing hpRNA complementary to S. sclerotiorum ABHYDROLASE-3 under the activity of a CaMV 35S promoter (AT35S::SS1G_01703RNAi herein referred to as AT1703). AT1703 A. thaliana was more tolerant to S. sclerotiorum infection with reduced lesion size, fungal load and ABHYDROLASE-3 transcript abundance compared to wild-type Col-0. Light microscopy of the infection process further identified reduced epidermal and mesophyll tissue degradation and vascular tissue colonization in AT1703 lines. Global RNA sequencing revealed enrichment of the SA-mediated SAR pathway and predictive gene regulatory network analysis predicted heat shock factor (HSFA4A, HSFA8) and TGA (TGA10) transcription factors as putative regulators of resistance in AT1703. Analysis of ABHYDROLASE-3 predicted interacting partners suggests this pathogenicity factor plays a role in the methionine salvage pathway which is necessary for successful polyamine biosynthesis. Taken together, these data demonstrate the utility of HIGS in slowing S. sclerotiorum infection and highlight the importance of the SA-mediated SAR pathway in contributing to successful defense against S. sclerotiorum.

HIGS of S. sclerotiorum ABHYDROLASE-3 slows infection in transgenic AT1703
Arabidopsis. hpRNA complementary to the ABHYDROLASE-3 gene coding sequence of S. sclerotiorum (SS1G_01703) was transformed into wild-type Col-0 A. thaliana under the control of the CaMV 35S promoter and propagated to the T3 generation. Three independent insertion lines were propagated to the T2 generation ( Supplementary  Fig. S1a), and our top performing line (AT1703.1, herein referred to as AT1703) was selected for RNA sequencing and further analysis at the T3 generation due to its significantly reduced lesion size and ABHYDROLASE-3 transcript reduction compared to wild-type Col-0 ( Supplementary Fig. S1b). Light microscopy of leaf cross sections using the chitin-targeting stain lactophenol blue showed no apparent differences in uninfected AT1703 and wild-type Col-0 leaves (Fig. 1a). However, at 2 dpi, wild-type Col-0 leaves show an increased abundance of S. sclerotiorum in both mesophyll and vascular tissues (Fig. 1b). At 3 dpi S. sclerotiorum shows further colonization of vascular tissue in wild-type Col-0 compared to AT1703 leaves, coupled with decreased levels of epidermal and mesophyll tissue degradation in AT1703 lines (Fig. 1c). At 1 dpi, no differences in lesion size or fungal abundance were observed (Fig. 1d,e), while at 2 dpi, S. sclerotiorum ABHYDROLASE-3 transcript levels showed a 73% reduction in AT1703 with no significant differences in lesion size or fungal load compared to wild-type Col-0 (Fig. 1f). However, by 3 dpi AT1703 showed a 48% reduction in lesion size, an 84% reduction in fungal load, and a 93% reduction in ABHYDROLASE-3 transcript accumulation compared to wild-type Col-0 plants (Fig. 1d,f). While this significant transcript reduction is present as early as 2 dpi, these data suggest a ~ 24-h lag phase where transcript abundance is reduced but insufficient to immediately slow infection (Fig. 1d,f).
Global RNA-sequencing reveals enriched gene activity of the salicylic acid-mediated SAR pathway in AT1703. To uncover the underlying gene expression patterns between AT1703 and wild-type Col-0 we performed global RNA sequencing on uninfected (0 dpi) leaves as well as directly at the site of infection at mid (2 dpi) and late (3 dpi) stages of S. sclerotiorum leaf infection. Hierarchical clustering of raw count expression values revealed distinct clusters forming between uninfected (0 dpi) and S. sclerotiorum infected tissues (Fig. 2). Genotypes also clustered in response to S. sclerotiorum infection compared to infection time points. Global gene activity showed no significant differences in the proportion of low, moderate or highly accumulated transcripts, with on average 20%, 21% and 58% of detected transcripts showing low, moderate and high-count levels respectively (Fig. 2). Further, total transcript detection between genotypes is similar, with an increase in  www.nature.com/scientificreports/ 1813 and 1348 detected transcripts in AT1703 compared to Col-0 at 2 and 3 dpi respectively due to an increased abundance of S. sclerotiorum transcripts in wild-type Col-0. Differential expression analysis identified shared and specifically up-regulated gene sets in response to S. sclerotiorum in AT1703 and wild-type Col-0 (Fig. 3a). For example, 335 and 235 genes were specifically upregulated at 2 and 3 dpi respectively, while 444 genes were up-regulated at both timepoints in AT1703. Further, 449 and 235 genes were down-regulated in AT1703 relative to Col-0 at 2 and 3 dpi respectively, and 351 genes were down-regulated at both timepoints (Supplementary Dataset S4). Next, to identify putative biological processes associated with these differentially expressed gene sets, we performed Gene ontology (GO) term enrichment (Fig. 3b). Data show the SA-mediated SAR pathway (p = 2.05 × 10 -27 ) and SA biosynthesis (p = 1.16 × 10 -31 ) as well as enrichment of the hypersensitive response (p = 3.07 × 10 -7 ) in gene sets up-regulated at 2 and 3 dpi in AT1703. The enrichment of SA biosynthesis, SAR and the hypersensitive response indicate a strong role of the SAR defense pathway in successful defense response and incompatible interaction (p = 1.28 × 10 -13 ) found in AT1703 lines. While SA biosynthesis and SAR demonstrate shared enrichment between AT1703 at 2 and 3 dpi, it is worth noting that statistically significant enrichment is also found specifically at 2 dpi, while no such enrichment is found for down-stream activated defense processes like the hypersensitive response and incompatible interaction (Fig. 3b). Further, 750 genes were up-regulated in response to S. sclerotiorum across both genotypes and timepoints (Fig. 3a). This shared gene set showed significant enrichment of PTI associated JA (p = 7.77 × 10 -34 ) and ET biosynthesis (p = 1.32 × 10 -49 ), induced systemic resistance (p = 6.63 × 10 -8 ) and PAMP induction of immune response (p = 1.71 × 10 -4 ) (Supplementary Dataset S1), suggesting that activity of these JA/ET-mediated defense pathways alone in wild-type Col-0 are insufficient to slow S. sclerotiorum infection without enrichment of SA-mediated SAR, providing additional evidence of the synergism of SA-mediated SAR and JA/ET-mediated ISR/PTI in mounting a successful defense against S. sclerotiorum. In contrast, enrichment of up-regulated genes specifically in wild-type Col-0 show association with programmed cell death and cell wall reinforcement, including leaf senescence (p = 1.97 × 10 -6 ), autophagy (p = 8.88 × 10 -6 ), cell wall modification (p = 2.61 × 10 -6 ) and organization (p = 2.75 × 10 -4 ), and lignin biosynthesis (p = 7.22 × 10 -7 ).
To explore potential regulators of immunity in AT1703 expressed throughout infection, we performed a transcription factor network analysis using SeqEnrich in our AT1703 up-regulated gene set in response to S. sclerotiorum. Here, we identified the TGA transcription factor TGA10, as well as the HSFs transcription factors HSFA4A and HSFA8, which are both predicted to regulate SA-signalling and SAR activity (Fig. 4a). Further, we explored differential expression between AT1703 and wild-type Col-0 in response to S. sclerotiorum in genes that have been previously described to interact with these transcription factors. Here, we found up-regulation of the TGA interacting ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), NONEXPRESSER OF PR GENES 1 (NPR1) and ISOCHORISMATE SYNTHASE 1 (ICS1) in AT1703 A. thaliana, as well as up-regulation of the HSFA4A interacting kinase MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) (Fig. 4b).

ABHYDROLASE-3 silencing influences expression of polyamine synthase genes in S. sclerotiorum.
While AT1703 demonstrates significant ABHYDROLASE-3 transcript reduction during S. sclerotiorum infection, we had yet to explore the influence of ABHYDROLASE-3 silencing on gene expression within the fungus itself. Three predicted interacting partners of ABHYDROLASE-3 (SS1G_02233, SS1G_14434, SS1G_03597) were identified in S. sclerotiorum using stringdb (https:// string-db. org/) (Fig. 5a). While SS1G_14434 and SS1G_03597 have yet to be significantly characterized, SS1G_02233 encodes a 5′-methylthioadenosine phosphorylase (MTAP) that prevents accumulation of the polyamine biosynthesis by-product S-methyl-5′-thioadenosine (MTA), inhibiting polyamine biosynthesis and ultimately fungal growth 31,32 . Here, all three predicted interacting partners showed significant transcript reduction in S. sclerotiorum that was infecting AT1703 compared to wildtype Col-0 at 2 and 3 dpi (Fig. 5b). Further, reduction in these predictive partners was consistent with our target gene ABHYDROLASE-3, showing at least a 50% increase in reduction from 2 to 3 dpi. To further support the role of ABHYDROLASE-3 in polyamine biosynthesis, we quantified expression of genes encoding the rate-limiting enzyme in polyamine biosynthesis ORNITHINE DECARBOXYLASE (ODC) as well as down-stream enzymes SPERMINE SYNTHASE and SPERMIDINE SYNTHASE. Transcript reduction was observed for all three genes in S. sclerotiorum challenging AT1703, suggesting a role of ABHYDROLASE-3 in polyamine biosynthesis during S. sclerotiorum infection.

AT1703 is not tolerant to the phytopathogen Botrytis cinerea. ABHYDROLASE-3 targeting
hpRNA expressed by AT1703 was carefully designed to not share 21-nt siRNAs with transcripts of related species including the closely related necrotrophic pathogen B. cinerea. To study whether AT1703 was also tolerant to B. cinerea, we next inoculated both wild-type Col-0 and AT1703 with B. cinerea spores (Fig. 6a). Here, no significant differences in lesion size (Fig. 6b), fungal load (Fig. 6c) or target transcript abundance (Fig. 6d) were found using the B. cinerea ABHYDROLASE-3 homolog BC1G_08022, suggesting no off-target effects in B.cinerea challenging AT1703.

Discussion
RNAi technology has the demonstrated ability to control pathogenic fungi in host plants through constitutively expressed dsRNA molecules in transgenic plants via host-induced gene silencing (HIGS). The constitutive expression of fungal-targeting dsRNAs allows for a durable means of protection against pathogen attack throughout the host plant lifecycle 16,33,34 . Protection of the plant relies on successful uptake and processing of dsRNA and processed siRNAs which can be generated in the host prior to uptake by the challenging pathogen 3 . Cross-kingdom trafficking of both dsRNAs and processed siRNA has been demonstrated across the host-pathogen interface in necrotrophic pathogens including S. sclerotiorum and the closely related B. cinerea, making these species ideal  www.nature.com/scientificreports/ Successful S. sclerotiorum infection involves the penetration and destruction of host epidermis and mesophyll tissue followed by colonization of the vasculature 28,37 . Once the vascular tissue has been colonized, infection is considered systemic, being able to cut off host nutrient supply while the fungus extends mycelia conveniently throughout host tissues. Susceptible genotypes have demonstrated preferential growth throughout the vascular tissue in Brassica species compared to respective tolerant lines 38 . While microscopy of S. sclerotiorum infected wild-type Col-0 and AT1703 demonstrates reduced S. sclerotiorum abundance in AT1703, we also find decreased levels of epidermal and mesophyll tissue degradation and reduced abundance of vascular tissue fungal colonization, thereby preventing systemic infection throughout the plant. Limiting systemic infection has significant implications on reducing yield losses from S. sclerotiorum infection. This was demonstrated by Wytinck et al. 16 , where transgenic B. napus lines expressing the same ABHYDROLASE-3 hpRNA construct against S. sclerotiorum show reduced vascular tissue colonization coupled with an increase in seed mass compared to untransformed B. napus post whole-plant S. sclerotiorum infection. As infection typically initiates on mature leaves before progressing into stem tissue through the vascular system 39,40 , it is critical to reduce vascular colonization directly at the site of infection, which can ultimately slow S. sclerotiorum progression throughout the host.
Target gene silencing in the pathogen is essential to slow the infection through HIGS. AT1703 plants demonstrate significant ABHYDROLASE-3 transcript reductions in S. sclerotiorum at 1 and 2 dpi, while reduced lesion size and fungal load are only identified at 3 dpi relative to wild-type Col-0 plants. Wytinck et al. 16 showed similar results in transgenic B. napus, with significant reductions in ABHYDROLASE-3 transcript abundance and fungal load observed at 1 and 2 dpi respectively. In contrast, prior HIGS studies targeting S. sclerotiorum  www.nature.com/scientificreports/ essential genes (TRX1, OA) detected both target transcript reduction and reduced lesion size at 2 dpi in host plants 33,41 , suggesting this delay to be specific to HIGS plants targeting ABHYDROLASE-3. These findings may be attributed to down-stream interactions of ABHYDROLASE-3 with other interacting partners. Reduced activity of ABHYDROLASE-3 in addition to regulators of polyamine biosynthesis are likely responsible for or at least contribute to the inhibition of fungal growth. However, further characterization of ABHYDROLASE-3 interacting partners and the polyamine biosynthesis pathway during ABHYDROLASE-3 silencing are required to better understand this delay. While the utility of HIGS in controlling S. sclerotiorum has been demonstrated in multiple host plants 16,33,34 , the underlying host response of these transgenic plants, as well as host-pathogen interactions in this system are less understood and dependent upon the specific gene being silenced. Here, global RNA sequencing and differential expression analyses provide insight into the response of AT1703 leaves to S. sclerotiorum directly at the site of infection. Moreover, the mode of pathogenicity of S. sclerotiorum has been recently debated, as emerging studies have suggested the presence of a brief biotrophic stage during early infection highlighted by the suppression of the SA-mediated SAR pathway 23,24 . SA biosynthesis in response to pathogen attack occurs through the isochorismate synthase (ICS) pathway, where ICS1 is essential for SA biosynthesis and successful SAR 42 . Downstream of SA accumulation, NPR1 activity is required for expression of pathogenesis-related (PR) genes involved in the SAR response through SA signal transduction 42,43 . AT1703 shows significant enrichment of the SA-mediated SAR pathway compared to wild-type Col-0, with enrichment of both ICS1 and NPR1 in response to S. sclerotiorum infection, suggesting an inability to suppress SAR while infecting AT1703 A. thaliana. Further, while NPR1 does not contain a DNA binding domain, it acts as a transcriptional cofactor, enhancing TGA transcription factor www.nature.com/scientificreports/ binding affinity to PR genes upon translocation into the nucleus in response to SA 44 . While multiple TGA transcription factors have been shown to interact with NPR1 in response to SA in A. thaliana 45 , TGA10 interaction has yet to be explored. Here, TGA10 was identified as the lone member of the TGA transcription factor family significantly enriched in AT1703 A. thaliana in response to S. sclerotiorum and was identified in our predictive transcription factor network to regulate SAR activity. Further, TGA10 has been demonstrated to be necessary for activation of reactive oxygen species (ROS) response in PTI in response to the bacterial PAMP flg22, where tga10 mutants were unable to activate flg22-response genes and showed reduced ROS production 46 , highlighting its importance in the activation of defense genes in response to pathogen attack. Our network analysis further identified two HSF transcription factors (HSFA4A, HSFA8) associated with the SA signalling pathway that are up-regulated in AT1703 in response to S. sclerotiorum. While HSFs have been shown to interact directly with EDS1, resulting in SA accumulation in response to pathogen attack 47,48 , similarly to TGA10, HSFA4A and HSFA8 have specifically been implicated in the host ROS response as well as activation of the host antioxidant system 49,50 . Activation of these HSFs can act through phosphorylation by MPK3 in response to stress, which also demonstrates significant up-regulation in AT1703 A. thaliana in response to S. sclerotiorum 49,51,52 . While these data suggest SAR activity is an essential component of a successful defense response against S. sclerotiorum infection, SAR alone is insufficient to protect the host, as quantitative disease resistance is complex and requires the coordination of multiple defense pathways and processes 53 . For example, SA-mediated SAR and JA/ET regulated ISR pathways exhibit synergistic effects in defense against necrotrophic pathogen attack 29 . This was demonstrated using npr1 knockout Arabidopsis mutants deficient in SA signalling, coi1 mutants deficient in JA signalling, and ein2 deficient in ET signalling, which all show hyper-susceptibility phenotypes due to the inactivity of SAR and ISR pathways 30 . Differential expression analysis not only identified enrichment of SAmediated SAR in AT1703, but also JA/ET biosynthesis and the ISR pathway which were significantly enriched in both AT1703 and wild-type Col-0 lines relative to their respective uninfected leaves, suggesting both defense pathways are essential in mounting a successful defense response against S. sclerotiorum.
To better understand the different defense responses to infection by wild-type Col-0 and AT1703 plants, we further explored defense processes enriched specifically in wild-type Col-0 and not AT1703. S. sclerotiorum demonstrates necrotrophic growth, especially at later stages of infection. Thus, the bi-products of gene activity belonging to autophagy and leaf senescence found in wild-type Col-0 can serve as nutrients for the progressing pathogen to continue growth as it feeds on necrotic host tissue 54,55 . With increased necrotic host tissue available to S. sclerotiorum in wild-type Col-0, host plants would require mechanical defenses through cell wall reinforcement and lignification in response to cell wall degrading enzymes being consistently secreted during actively progressing infection 56 . Lignification in response to S. sclerotiorum has previously been described by Höch et al. 56 , where genes associated with lignin biosynthesis showed significantly increased expression levels in susceptible B. napus lines compared to moderately resistant lines, specifically during mid to late stages of infection. These findings agree with our data where we find enrichment of lignin biosynthesis and cell wall reinforcement in wild-type Col-0 lines compared to AT1703 throughout S. sclerotiorum infection.
While we have examined the influence ABHYDROLASE-3 silencing has on the host response, there is also a need to explore differences within the pathogen and the effect that ABHYDROLASE-3 silencing has at the molecular level. ABHYDROLASE-3 is annotated as a tRNA synthetase but also predicted to be involved in aflatoxin biosynthesis. However, this gene has yet to be further characterized beyond its ability to slow S. sclerotiorum infection through SIGS and HIGS RNAi treatments 15,16 . Using the stringdb (https:// string-db. org/) protein-protein interaction network database, we identified three predictive interacting partners of ABHY-DROLASE-3 (SS1G_14434, SS1G_03597 and SS1G_02233). Expression levels of ABHYDROLASE-3 and these predicted interacting partners in S. sclerotiorum were coordinately reduced in transgenic AT1703 compared to wild-type Col-0. While SS1G_14434 and SS1G_03597 have yet to be functionally characterized in S. sclerotiorum, SS1G_02233 encodes MTAP, which plays a critical role in cleaving and processing accumulated MTA, a by-product of polyamine biosynthesis 31,32 . Buildup of MTA through inactivity of MTAP ultimately results in the inhibition of polyamine biosynthesis 31,32 . This in turn can have significant impacts on fungal growth, as the three most widely distributed polyamines, putrescine, spermidine and spermine have been shown to be necessary for successful growth and cell differentiation 32,57 . The ornithine decarboxylase (SS1G_05468) is an essential enzyme of polyamine biosynthesis, converting ornithine into the polyamine putrescine 32,57 . Once ornithine is converted into putrescine, there are further down-stream synthase genes necessary for conversion into both spermine and spermidine. These enzymes include spermine synthase (SS1G_12906) to convert putrescine into spermine, and spermidine synthase (SS1G_10282) to convert spermine into spermidine. Our RT-qPCR analysis of these essential polyamine synthesis genes shows transcript reduction during S. sclerotiorum infection of AT1703 plants compared to their wild type susceptible counterparts, suggesting a potential interaction between ABHYDROLASE-3 and the identified predictive partners of the polyamine biosynthesis pathway. Further, as putrescine can also be produced through spermine and spermidine catabolism, expression of rate limiting enzymes SSAT and PAO can also be explored to improve our understanding of ABHYDROLASE-3 influence on polyamine metabolism 32 . While the potential direct or indirect interaction of ABHYDROLASE-3 and these predictive interacting partners is still unclear, these data provide some preliminary insights of the role ABHY-DROLASE-3 plays in S. sclerotiorum pathogenicity and further identifies a group of promising RNAi candidate genes through the polyamine synthesis pathway.
One benefit of RNAi and HIGS technology in crop protection is the ability to design pathogen specific molecules that are ineffective against potentially beneficial species in the ecosystem 4,58 . This requires the careful design of target sequences within genes, as dsRNA molecules containing as few as three overlapping 21-mer nucleic acid sequences have demonstrated the ability to significantly reduce off-target transcripts 59 . B. cinerea serves as an ideal candidate to study these potential off-target effects in controlling S. sclerotiorum given both pathogens share similar hosts and disease cycles in addition to having a high level of sequence homology 60 15 showed that dsRNA targeting the ABHYDROLASE-3 B. cinerea homolog BC1G_08022 was able to reduce B. cinerea lesion size, fungal load and BC1G_08022 transcript abundance when applied to B. napus leaves prior to B. cinerea inoculation. While BC1G_08022 is capable of slowing B. cinerea infection when targeted, our dsRNA region targeting S. sclerotiorum ABHYDROLASE-3 shows no 21-mer overlapping regions with off-target species (Supplementary Dataset S2) and AT1703 A. thaliana challenged with the closely related necrotrophic fungal pathogen B. cinerea shows no significant differences in lesion size, fungal load or BC1G_08022 transcript reduction. Taken together, these data demonstrate the efficacy of HIGS as an alternative control measure in slowing S. sclerotiorum infection through targeted silencing of S. sclerotiorum ABHYDROLASE-3. Reduction of ABHY-DROLASE-3 in S. sclerotiorum provides A. thaliana the ability to increase innate defense responses through SA-mediated SAR at the global mRNA level. The identification of co-silenced ABHYDROLASE-3 interacting partners further uncovers a promising group of candidates for RNAi control against fungal pathogens. Finally, with ongoing concerns of potential off-target effects in RNA-based applications, we demonstrate the specificity of HIGS targeting S. sclerotiorum in AT1703 to a closely related fungus. Taken together, we provide evidence that HIGS applications can be used as an effective and sustainable strategy in crop improvement against necrotrophic pathogens. Initially, gene fragments were ligated into the pENTR4 vector before insertion into the pHellsgate8 destination vector. To confirm insert identity, entry vectors were sequenced at the Centre for Applied Genomics in Toronto, Ontario. Inserts were then recombined into the destination vector using a Gateway LR clonase reaction (ThermoFisher Scientific) following the manufacturer's instructions, with the modification of adding 4:1 entry vector to destination vector ratio according to Wytinck et al. 16 . A cold shock treatment was used to transform Agrobacterium. Successful transformants were selected using colony PCR with XhoI and XbaI separate restriction enzyme digestions. Cultures of Agrobacterium were grown to OD 1.6-2.0, pelleted using centrifugation, and re-suspended in MS media and 0.001% Silwet L-77. To transform A. thaliana, seeds were initially sterilized using alternating washes of 70% and 95% ethanol then placed on Murashige and Skoog (MS) media (Sigma Aldrich). Seeds were vernalized for three days at 4 °C prior to being grown in a constant light incubator. Upon formation of first leaves A. thaliana seedlings were transplanted into Sunshine Mix #1 and grown to maturity at 22 °C. Mature flowering A. thaliana plants were dipped into the Agrobacterium culture and kept in high humidity conditions for two days. Floral dips were repeated five times before seeds were dried and harvested. Transformants were selected using MS and 150 μg/mL kanamycin media 62 . PCR was used to confirm the presence of the S. sclerotiorum gene using the same primers used for cloning within the plant (Supplementary Dataset S3). Three confirmed independently transformed A. thaliana lines were grown to the T2 generation and tested for their ability to silence ABHYDROLASE-3 transcripts and slow S. sclerotiorum infection. The top performing line (AT1703.1) was subsequently selected for further RNA sequencing analysis.

Methods
Arabidopsis thaliana infection assays. Sclerotinia sclerotiorum ascospores were collected at the Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB, Canada and stored at 4 °C in desiccant in the dark according to Ref. 15 . S. sclerotiorum ascospore inoculum was made by suspending a 1 × 10 6 spore/mL concentration of spores in a potato dextrose broth (PDB) and peptone solution (24 g PDB, 10 g peptone, 1 L water). Using a pipette, 10 µL of the solution was transferred onto mature transgenic and wildtype Col-0 A. thaliana leaves at a n = 15 leaves per treatment. Plants were stored in growth chambers at room temperature (21.0 °C), with plant trays being sealed with lids to maintain high levels of humidity for three days, allowing for infection progression in planta. At 2-and 3-days post inoculation (dpi), lesion area was quantified using ImageJ software and leaves were collected for RNA sequencing, fungal load, and transcript abundance experiments. Fifteen infection sites were quantified per treatment while three leaves were collected per bio-replicate with three biological replicates collected per treatment. Botrytis cinerea infections were performed using ascospores from the Saint-Jean sur Richelieu Research and Development Centre, Agriculture and Agri-Food Canada, QC, Canada following the same protocol for S. sclerotiorum ascospore inoculations. Raw count data was generated using featureCounts 67 and inputted into pvclust (https:// cran.r-proje ct. org/ web/ packa ges/ pvclu st/ index. html) for hierarchical clustering analysis with a detection cutoff ≥ 1. Detected transcripts were subsequently sorted into low (1 ≥ 5), moderate (5 > 25), or high (≥ 25) abundance levels. Further, raw count data was processed in DESeq2 for differential expression analysis. Here, differentially expressed genes (DEGs) were identified between pairwise comparisons at a p-value cutoff of p < 0.0001. GO enrichment of identified DEGs was performed using SeqEnrich 68 with terms being considered significantly enriched at p ≤ 0.001 according to software specifications. GO heatmap visualization was performed using the conditional formatting function in Excel. GO summary tables can be found in Supplementary Dataset S1. Predictive transcription factor networks were generated using SeqEnrich (Supplementary Dataset S5) with DEG gene lists being used as input.